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SUMMARY The hepatitis E virus (HEV) is a major cause of acute hepatitis globally. Genotypes 1 and 2 (G1 and G2) are obligate human pathogens transmitted faeco-orally, leading to epidemics in developing countries. In contrast, genotypes 3 and 4 (G3 and G4) have a wider host range, including humans, but are primarily porcine viruses and are transmitted from animals to humans as a food-borne zoonosis when meat from an infected animal is consumed. HEV is increasingly recognised as a problem in developed countries, including countries in Europe. G3 HEV is now the most common cause of acute viral hepatitis in the UK and cases continue to rise. The majority of these infections are acquired within the UK and thought to be from insufficiently cooked meat, predominantly processed pork meat. Previously thought to only cause self-limiting disease, HEV infection can persist in immunosuppressed patients, which may lead to chronic hepatitis and the rapid development of cirrhosis. Of particular interest to the transfusion community has been the possibility of transfusion-transmitted HEV, which has been reported from countries classically considered HEV-endemic but also non-endemic countries in Europe and Japan. This has prompted some countries to introduce screening for HEV in blood donations.

The hepatitis E virus (HEV) is a major cause of acute hepatitis globally. Genotypes 1 and 2 (G1 and G2) are obligate human pathogens transmitted faeco-orally, leading to epidemics in developing countries. In contrast, genotypes 3 and 4 (G3 and G4) have a wider host range, including humans, but are primarily porcine viruses and are transmitted from animals to humans as a food-borne zoonosis when meat from an infected animal is consumed. HEV is increasingly recognised as a problem in developed countries, including countries in Europe. G3 HEV is now the most common cause of acute viral hepatitis in the UK and cases continue to rise. The majority of these infections are acquired within the UK and thought to be from insufficiently cooked meat, predominantly processed pork meat. Previously thought to only cause self-limiting disease, HEV infection can persist in immunosuppressed patients, which may lead to chronic hepatitis and the rapid development of cirrhosis. Of particular interest to the transfusion community has been the possibility of transfusion-transmitted HEV, which has been reported from countries classically considered HEV-endemic but also non-endemic countries in Europe and Japan. This has prompted some countries to introduce screening for HEV in blood donations.

Due to the relatively recent emergence of the human T-lymphotropic and the human immunodeficiency viruses, enthusiasm for the identification of novel viruses, especially retroviruses, with pathogenic potential in humans, remains high. Novel technologies are now available with the ability to search for unknown viruses, such as gene arrays and new generation sequencing of tissue and other samples. In 2006, chip technology identified a novel retrovirus in human prostate cancer (PCa) tissue samples. Due to close homology to a mouse retrovirus, the virus was named xenotropic murine leukaemia virus-related virus (XMRV). Ever since the initial disease association with PCa, XMRV has stirred a lot of attention and concern worldwide for the medical community, public health officials and in particular global transfusion services. Public response, in this new era of electronic communication and advocacy was rapid, wide and unprecedented. In this review, we outline the course of biomedical research efforts that were put forward internationally in the process of determining the risk to the human population, the response of the blood banking community and review the current state of knowledge of xenotropic murine retroviruses. Although XMRV is no longer regarded as an infection of humans, a lesson was learnt in modern virology that holds deeper implications for biomedical research, particularly stem cell generation and transplantation practices.

During viral assembly, viral proteins are released into plasma and can be used to infer viral load. The Architect hepatitis C virus (HCV) core antigen (Ag) assay is a potential alternative to HCV RNA quantification for measuring response to therapy and predicting an end of treatment viral response (EOTR). The HCVp22Ag assay was used to infer viral load in 68 window RNA-containing samples and in 284 samples from baseline to week 14 of ribavirin/interferon treatment in 23 patients with EOTR including three who relapsed, 20 not achieving EOTR and 11 controls. HCV Ag and RNA correlated well (r = 0.86) with linear dose responses on dilution. In patients on therapy and control patients, plasma HCV antigen was detected in 51 of 54 with an interpolated LOD cut off between 10(3) and 10(4) RNA IU/mL. Plasma HCV antigenaemia and plasma RNA levels were significantly different in EOTR from non-EOTR patients at 3 days after treatment start and all times thereafter. Positive and negative EOTR predictive values for HCV RNA2 log drop and HCV Ag loss at 12 weeks were 70% and 74%, 85% and 93% respectively. HCV Ag reactivity has a linear dose response independent of genotype and correlates well with HCV RNA. The failure to clear HCV Ag is as accurate as the failure to clear HCV RNA at twelve weeks into therapy in predicting the likelihood of failure to achieve EOTR. HCV Ag potentially offers a convenient alternative to RNA measurement for defining a futility flag in HCV therapy.

South London Specialist Virology Centre, King’s College Hospital, Denmark Hill, London SE5 9RS, United Kingdom Virus Reference Department, Microbiology Services Colindale, 61 Colindale Avenue, London NW9 5HT, United Kingdom Department of Histopathology, King’s College Hospital, Denmark Hill, London SE5 9RS, United Kingdom Department of HIV/Genitourinary Medicine, King’s College Hospital, Denmark Hill, London SE5 9RS, United Kingdom Department of Haematological Medicine, King’s College Hospital, Denmark Hill, London SE5 9RS, United Kingdom

During the 1918, pandemic blood components were successfully used to treat severe influenza pneumonia. A Proof of Principle trial investigating the clinical benefit of convalescent plasma was proposed in the 2009 H1N1v epidemic with the aim of screening donors for high titre antibody in order to stockpile plasma packs to be used for treatment for severe pneumonia.Serum samples were collected from donors. IgG antibody capture format enzyme-linked immunoassays using recombinant proteins (GACELISAs) were compared with microneutralization (MN) and haemagglutination inhibition (HAI). The influence of age and history of influenza-like illness (ILI) on the detection of high titre antibody was examined.1598 unselected donor sera collected in October and December 2009 were tested by HAI. The HAI and demographic data defined a possible strategy for selective donor screening. One of the GACELISAs was highly specific for recent infection but showed lower sensitivity than HAI.During the 2009 pandemic screening 17- to 30-year-old donors by HAI delivered around 10% with high antibody levels. The ELISA using a short recombinant H1N1v HA detected fewer reactives but was more specific for high titre antibody (≥1:256). Screening strategies are proposed based on using HAI on serum or GACELISA on plasma.

A community survey of human T cell lymphotropic virus type I (HTLV-I) in Montserrat, West Indies, identified 22 instances in which 2 HTLV-I-seropositive adults lived within 60 m of each other (close pairs), compared with 7.8 expected (P < .001). Five of these close pairs were mother-offspring or husband-wife. The remaining 17 pairs were of unrelated members in separate households. The percentages of male-female (41%), female-female (41%), and male-male (18%) types in these 17 pairs were very similar to those among the 1377 similarly defined pairs in which neither or only 1 member was seropositive, affording no support for extramarital heterosexual activity as an explanation for the clustering observed. Thus, the demography of HTLV-I was not accounted for completely by sexual and mother-to-offspring transmission. The predominance of clustering of unrelated HTLV-I-seropositive individuals in locations with high mosquito infestation raised the possibility of sporadic transmission of HTLV-I by hematophagous insects.

The risk of hepatitis E virus (HEV) to blood safety remains unknown in England. Reports of persistent HEV infection with serious disease sequelae indicate that transfusion transmitted HEV is not a trivial disease in immunosuppressed patients.Samples from unselected blood donors and donors with a history of jaundice were tested for HEV antibody and RNA.Overall, 10% of the donor sera were anti-HEV IgG reactive. Four of the donor samples were anti-HEV IgM reactive but HEV RNA negative.There is evidence of probable recent HEV infections in donors with a predicted attack rate of 2.8%.

Thirteen patients with AIDS or ARC on continuous zidovudine therapy (800–1200 mg daily), who developed severe anaemia (median Hb 7.9g dl−1 range 4.9–9.4) after 66 days of treatment (median, range 35–258) were then treated with an intermittent regimen in an open study. The regimen consisted of cycles of 4 weeks on treatment (600–100 mg daily) followed by 2 weeks off. Intermittent treatment was tolerated for a significantly longer period of time (median 257 days range 72–603) and a higher cumulative does ( P < 0.001). Four patients discontinued intermittent therapy in the period of follow-up, three due to bone marrow suppression. Eight of 13 patients needed red cell transfusions on continuous therapy compared to only one on the intermittent regimen. On intermittent therapy median haemoglobin concentration and neutrophil counts remained stable (Hb median 11.9g dl−1 range 9.1–14.8, neut. median 1.7 × 109I−1 range 0.6–4.3). Ten patients had detectable levels of P24 antigen which decreased from a median of 128 pg ml−1 (range 15–242) to 24 pg ml−1 (range 0–53) on continuous therapy. Subsequently, in eight of these patients on the intermittent regimen, levels of P24 Ag remained below 50% of initial pretreatment values (median 28 pg ml−1 range 0–178) or below 50 pg ml−1.

Background and Objectives Certain patient ethnic groups may require blood components from donors under-represented in the UK donor population. Selective recruitment of Afro-Caribbean donors is therefore necessary but was considered to pose an increased risk of human T-cell leukaemia/lymphoma virus (HTLV) infection. To assess this a seroprevalence study of HTLV was undertaken in Afro-Caribbean and Caucasian donors.Materials and Methods Sera from 1100 Afro-Caribbean and 1100 Caucasian donors were tested for antibody to HTLV. Reactive samples were confirmed for specificity using an algorithm comprising two additional assays and polymerase chain reaction (PCR) where possible.Results Six Afro-Caribbean donors (0.55%) were considered to be infected with HTLV I.Conclusion Donor selection in this case caused a significantly elevated prevalence of HTLV infection and serves as a warning of the need for care in the design policies for selective donor recruitment.

SUMMARYBetween March and June 2008, 12 cases of hepatitis A were notified in Winchester. Cases were from a primary school and a nursery school with no direct linkage. Hepatitis A virus (HAV) RNA sequenced from nine cases confirmed the strain in both schools to be identical. The outbreak could have affected three other schools and a maternity unit and was controlled by immunization and screening of neonates in the maternity unit by dried blood spots. No neonates were infected and no further cases were reported until 5 months later when the index case's mother became infected with same strain of virus associated with the outbreak despite vaccination. Neither the source of the outbreak or the subsequent infection of the index case's mother was identified; however, with the timing of the cases continued transmission in the community by children with asymptomatic infection or a recurrent source cannot be ruled out.

SUMMARYThe cross-reaction of HHV6 antibody with that to the other herpesviruses was studied in 96 blood donors whose sera were tested for IgG antibody to human herpesvirus type 6 (HHV6), cytomegalovirus (CMV), Epstein–Barr virus (EBV), varicella zostervirus (VZV) and herpes simplex virus (HSV). No correlation was found between IgG antibody to HHV6 and that to any of the other herpesviruses in these individuals. Antibodies to HHV6 and CMV were measured in patients undergoing documented serological responses to HHV6. Eleven cases of primary HHV6 infection associated with roseola infantum in babies, 1 of whom suffered from gastroenteritis as well as pyrexia and rash, are reported. Three cases of HHV6 reactivation, 1 in a 3-year-old child and 2 in adults, 1 of whom simultaneously underwent a primary CMV infection are also reported. Our results suggest that indirect immunofluorescence is a specific way of measuring HHV6 antibody, that HHV6 IgG and IgM can be detected in the absence of antibody to CMV and that HHV6 IgM is present both in primary HHV6 infections and in reactivations.

To date, 11 HIV-1 M group clades, A to K, have been characterized, displaying different distributions, prevalences, and biological properties. Approximately 90% of new HIV-1 infections occur in developing countries, including the Caribbean. However, information on HIV-1 subtypes from this region is limited. We report subtype characterization of viruses from 71 individuals, obtained during the period 2000–2002. RNA from the pol region was sequenced, generating data on subtype and drug resistance associated mutations for 71 specimens from 9 countries. Sixty-seven (94.4%) sequences were classified as clade B, three (4.2%) as D/B, and one (1.4%) as clade C. Numerous polymorphisms were observed, including some associated with drug resistance, but not signifying exposure to chemotherapy. This study adds to our knowledge of HIV-1 clades in the Caribbean, and indicates possibilities for monitoring HIV-1 chemotherapy.

In order to reduce the potential for transmission of hepatitis C virus (HCV) from an RNA-positive, anti-HCV-negative blood donation, the National Blood Service (NBS) introduced nucleic acid amplification technology (NAT) testing for HCV in England and Wales. The objective of this study was to develop an automated assay using commercial components for the detection of HCV RNA in blood donations for transfusion.The Qiagen QIAamp 96 'Viral RNA' and 'Virus' BioRobot kits for HCV RNA extraction, and the Roche COBAS HCV Amplicor v2.0 and AmpliScreen v2.0 assays for polymerase chain reaction (PCR) amplification and detection, were investigated.QIAamp technology and the BioRobot 9604 allow automation of the viral RNA extraction process. By combining the automated silica-membrane based QIAamp 96 Virus extraction and automated reverse transcription-polymerase chain reaction (RT-PCR) set-up with COBAS HCV AmpliScreen v2.0 amplification and detection it is possible to achieve a 95% detection level for HCV of 12.8 IU/ml. Cross-contamination studies have shown that use of the BioRobot 9604 does not pose a detectable contamination risk. Between 1999 and 2001, approximately 6.8 x 106 donations were tested in England and Wales, of which only four were found to contain RNA without anti-HCV.This combination of methods results in an assay with a high sample throughput, little 'hands-on' time and fast turnaround time that is also sufficiently sensitive to allow testing of pools of up to 96 samples at a time. These methods have been successfully introduced into routine use within the NBS for release of blood components with a shelf-life of longer than 24 h.

Certain patient ethnic groups may require blood components from donors under-represented in the UK donor population. Selective recruitment of Afro-Caribbean donors is therefore necessary but was considered to pose an increased risk of human T-cell leukaemia/lymphoma virus (HTLV) infection. To assess this a seroprevalence study of HTLV was undertaken in Afro-Caribbean and Caucasian donors.Sera from 1100 Afro-Caribbean and 1100 Caucasian donors were tested for antibody to HTLV. Reactive samples were confirmed for specificity using an algorithm comprising two additional assays and polymerase chain reaction (PCR) where possible.Six Afro-Caribbean donors (0.55%) were considered to be infected with HTLV I.Donor selection in this case caused a significantly elevated prevalence of HTLV infection and serves as a warning of the need for care in the design of policies for selective donor recruitment.

Pre-emptive treatment strategies based on sensitive screening for cytomegalovirus (CMV) infection up to day +100 after allogeneic transplantation have been shown to reduce the incidence of CMV disease during the period of surveillance. However, the use of ganciclovir has been associated with delays in immune reconstitution and an increased incidence of late CMV disease after day +100. In the present study, 81 patients undergoing allogeneic transplantation received polymerase chain reaction (PCR)-guided pre-emptive therapy based on detection of CMV DNA by PCR on 2 consecutive weeks up to day +180. Thirty-three of the 52 high-risk patients (CMV-seropositive donor or recipient) received a total of 45 treatment episodes up to day +100. Three of these patients (5.7%) developed CMV disease, with one fatality. Twelve of the surviving 44 high-risk patients (27%) required pre-emptive treatment between days +101 and +192, but none of these patients developed late CMV disease with a median follow-up of 402 d (range 117-952 d). Antiviral therapy was stopped after a single negative PCR result with no subsequent episodes of CMV disease while patients remained off antiviral treatment. As all initial episodes of CMV DNA detection occurred within 60 d of transplantation, it may be possible to discontinue monitoring beyond day +100 in patients who have remained CMV PCR negative before this. Thus, we have confirmed that PCR-guided pre-emptive therapy results in a low incidence of CMV disease before day +100 and that discontinuing treatment on the basis of viral clearance as determined by CMV PCR appears to be safe practice. In addition, we have observed no episodes of late CMV disease with an extension of surveillance to 26 weeks.

To determine the stability of hepatitis C virus (HCV) RNA during transport and storage of blood samples from donors, prior to screening for HCV by nucleic acid amplification technology.Various blood and plasma sample types were stored for up to 120 h at different temperatures and the HCV RNA level was measured using an in house quantitative reverse transcription-polymerase chain reaction.No decline in HCV RNA level was observed after 72 h of storage of whole blood at 4 degrees C in EDTA tubes (Greiner) and Plasma Preparation Tubes (PPT; Becton Dickinson), while insignificant declines of 0.2 log10 and 0. 25 log10 occurred at 25 degrees C after 72 h in the EDTA tubes and PPT tubes, respectively. When whole blood was stored with mixed anticoagulants CPDA-1 and EDTA for up to 120 h, no decline in HCV RNA level was observed at 4 degrees C and 25 degrees C, while a significant decline of 0.37 log10 occurred at 37 degrees C after 120 h. The temperature during transportation was investigated with a 12-hour period at 25 degrees C and 37 degrees C before storage at 4 degrees C for 108 h. Neither temperature resulted in any loss of HCV RNA in comparison with 120 h of storage at 4 degrees C.Whole blood anticoagulated with EDTA or CPDA-1/EDTA may be stored at up to 25 degrees C (room temperature) for up to 5 days without any significant loss in plasma HCV RNA level.

Ideally HIV antibody tests have to be both extremely sensitive and able to recognize all known HIV subtypes. Three patients whose sera failed to react with a synthetic oligopeptide-based HIV antibody test are described in this report. The patients were a Pakistani male infected recently, an Australian male infected for several years, and a Ugandan woman with AIDS. The presence of anti-HIV antibodies was confirmed by means of a standard algorithm with different assay formats. All three sera failed to react in one antiglobulin enzyme-linked immunosorbent assay (ELISA) (Bioelisa HIV-1+2, Biokit SA). No single underlying reason could be identified for the assay failure in the three cases. The first patient, probably infected recently when first tested, was strongly positive by the same assay a year later, confirming the relative insensitivity of oligopeptide assays reported previously for detecting the early antibody response. The other two patients appear to have been infected for several years. Although unlikely to have been infected with a non-clade B virus, the sample from patient 2 lacked detectable antibody to the transmembrane glycoprotein (gp41), the site of the synthetic oligopeptides. Patient 3, of Ugandan origin, was found to be infected with a non-clade B virus. Although her serum reacted strongly to subtype B gp41 in Western blot, it failed to react in the antiglobulin ELISA. Since there appears to be no single common explanation for these three failures there is little opportunity to identify prospectively those situations where testing using assays employing synthetic oligopeptides on the solid phase is likely to fail.

Blood donor screening for antibody to hepatitis B core antigen (anti-HBc) implemented in some countries as a surrogate marker for non-A, non-B hepatitis has been superseded by anti-HCV screening. To assess the value of anti-HBc screening for the detection of hepatitis B surface antigen-negative blood donations that might contain infectious HBV, HBV genomic detection and recipient testing were used. Blood donations were screened and confirmed by multiple anti-HBc assays. Donations containing isolated anti-HBc and those with anti-hepatitis B surface antigen (anti-HBs) level0.1 IU/ml were tested for the presence of HBV DNA. Recipients of previous donations from the corresponding donors during the previous 5 years were traced and tested for markers of HBV infection. Of 103 869 donations screened, 586 (0.56%) were anti-HBc positive, two of which contained HBsAg, and 413 (0.4%) had protective (/= 0.1 IU/ml) levels of anti-HBs. Anti-HBs0.1 IU/ml was found in 102 of these donations (0.1%) and isolated anti-HBc in 69 (0.07%). No donations with isolated anti-HBc were HBV DNA confirmed positive. Of 278 recipients of previous donations from 171 donors at risk of HBV carriage, 12 had markers of HBV infection. Six recipients had other identified risk factors. An association with blood transfusion was considered probable in two and possible in four recipients. None of the six corresponding donors had detectable HBV DNA 6-40 months after the implicated donation. The frequency of HBV transmission by chronic carriers negative for hepatitis B surface antigen was estimated in this study to be 1 in 52,000 donations (CI 0.3-7.8/100,000) from HBsAg-negative donors. Such HBV infectious donations may not be detected by DNA amplification.

To investigate the infectivity for hepatitis C virus (HCV) of intravenous anti-D immunoglobulin batches manufactured in Ireland between 1991 and 1994.Women who had received anti-D manufactured between 1991 and 1994 were screened for serological markers of HCV infection and for the presence of HCV RNA by RT-PCR amplification and virus genotyping.44 women exposed to anti-D manufactured between 1991 and 1994 were polymerase chain reaction positive for HCV RNA, 19 of whom were infected with genotype 3a virus shown by phylogenetic analysis of the NS5B gene to be closely related to that from the single implicated donor.Anti-D manufactured in 1991-1994 transmitted infection of HCV genotype 3a. The prevalence of HCV-specific antibody in anti-D recipients was relatively low (0.59%), consistent with the low level of virus RNA in these anti-D batches.

Replication of HIV introduces errors into the genome which are responsible for conferring a growth advantage over wildtype virus when drugs such as zidovudine (ZDV) exert a selective pressure. The molecular basis for HIV-1 resistance to ZDV has been mapped to codons 41, 67, 70, 215 and 219 of the reverse transcriptase gene both in vitro and in clinical samples of blood. This study has investigated the relationship between the quantitative prevalence of ZDV resistance in multiple organs of the same individual. Proviral HIV-1 load was measured by quantitative-competitive PCR in 90 samples from organs of 11 patients dying with AIDS. Nine of these patients had been prescribed zidovudine. The distribution of wildtype and mutant sequences at the positions 41, 67, 70, 215 and 219 of the reverse transcriptase was assessed using a point mutation assay. The results showed that the highest proviral loads were predominately found in lymph node, spleen and lung and there was a significant association between viral load and resistance to ZDV (P=0.008). Inter-organ distribution of wildtype and mutant sequences at codons 41, 67, 70, 215 and 219 was frequently not uniform and in some patients differed markedly between the lymphoreticular system and other organs. These results demonstrate that treatment of HIV-1 infection with zidovudine does not exert uniform selective pressures in multiple organs. These findings have implications for the interpretation of resistance data and design of treatment strategies for HIV, arguing in particular that alterations in therapeutic regimens should consider the likelihood of different resistance patterns being present in multiple sites within the same individual.

In a series of fourteen patients with adult T-cell lymphoma-leukaemia (ATLL) in Brazil the main features were lymphadenopathy, hepatosplenomegaly, hypercalcaemia, and high leucocyte counts, with abnormal lymphoid cells which had irregular nuclei. The series included the youngest patient with ATLL so far (18 months). Analysis with monoclonal antibodies showed a mature T-cell phenotype (CD4+, CD8-). Antibodies to HTLV-I and/or integration of HTLV-I proviral DNA were found in eleven patients. In the other three HTLV-I DNA could not be demonstrated even by means of the polymerase chain reaction; they therefore had HTLV-I-negative ATLL. This report of ATLL in Brazil corroborates serological reports that HTLV-I may be endemic in some parts of that country. Follow-up studies are required to identify precisely the main route of transmission of HTLV-I in South America and the risk factors for the development of ATLL in carriers.

The expression of hepatitis B virus antigens was studied by double staining liver tissue with appropriate antisera and correlated with serum hepatitis B viral DNA and histology in 28 patients with disease related to chronic hepatitis B virus infection. The cellular localization of hepatitis B core and hepatitis B e antigens generally coincided, but there were important differences at a subcellular level. Thus, hepatitis B e antigen was detected in nuclei and/or cytoplasm but strong cytoplasmic hepatitis B e antigen was associated with a high serum hepatitis B viral DNA (P = 0.0017) but not with active liver disease. Hepatitis B core antigen could also be detected in nuclei and/or cytoplasm, but strong cytoplasmic hepatitis B core antigen expression, exceeding that of hepatitis B e antigen, was associated with active liver disease (P = 0.041) and not with serum hepatitis B virus DNA. The proportion of hepatocytes expressing hepatitis B surface antigen correlated inversely with the serum titer (P = 0.0017), whereas hepatitis B surface and nucleocapsid antigens were usually expressed independently. The data support the hypothesis that cytoplasmic hepatitis B core antigen and not hepatitis B e antigen is the target for immune system-mediated cytolysis of hepatocytes. Cytoplasmic hepatitis B e antigen is not associated with liver damage but is instead associated with high levels of hepatitis B virus replication.

SummaryRubella vaccination histories were taken from 333 young women working in the head office of a retail organization: 29% said they had had vaccine and 47% said they had not. The remainder did not know. Forty-six per cent of those ≤ 25 years old (who should have been offered vaccine at school), and 6% of those > 25 years old, said they had been vaccinated. When screened for immunity to rubella by radial haemolysis (RH) 3% had a low level of antibody (< 15 i.u./ml) and 11% had no antibody. After immunization with Cendevax the specific rubella IgM response was measured by an IgM antibody capture radioimmunassay (MACRIA). It was only detectable in the group without RH antibody, and was present in 26/31 of them. The Ig M response to Cendevax was strongest in specimens taken 20–39 days after immunization, but in 10 out of 11 cases tested was still present at around 71 days. The specific Ig M responses to Cendevax were very similar to those in women given Almevax in an earlier study, when measured in parallel tests.Taking both vaccines together, specific Ig M was present in 35 out of 36 vaccinees without pre-existing antibody tested between 40 and 77 days post-immunization. Detection of specific IgM by MACRIA would therefore be an effective means of determining susceptibility retrospectively in rubella vaccinees found to be pregnant.

The sensitivity of nine commercial assays, Western blot, and a newly developed monoclonal antibody-based assay for antibody to human T lymphotropic virus type III/lymphoadenopathy associated virus (anti-HTLV III/LAV) were evaluated using a panel of mainly weak-positive sera. In tests on 20 sera three commercial assays and a monoclonal-based assay, representing two different solid-phase methodologies, were found to be more sensitive than Western blot. Our findings suggest that Western blot cannot be depended upon as the sole confirmatory test for anti-HTLV III/LAV. The continued use of the more sensitive enzyme-linked immunosorbent assays (ELISAs), particularly those of dissimilar methodology, would be a more valid approach to confirmatory testing.

SUMMARYEight sera from 125 cases of infectious mononucleosis (IM) were reactive for rubella-specific IgM in an M-antibody capture radioimmunoassay. The reactivity of individual sera varied depending upon the source of the rubella antigen used in the assay. One serum gave strongly positive results with some rubella haemagglutinating antigens but negative results with others and may have contained an IgM antibody which was capable of distinguishing between strains of rubella virus.If the diagnosis of rubella is based solely on detection in solid-phase immunoassay of rubella-specific IgM, IM should be excluded.

SUMMARYHybridomas secreting monoclonal antibodies to HBsAg and HBcAg were prepared from immunized mice. An antibody capture radioimmunoassay was used to detect and select appropriate hybrids for propagation and cloning. The advantages of this assay were discussed. The resulting monoclonal antibodies were compared with conventional polyclonal antisera for the detection of virus antigens in liver tissue and found to give excellent results.

Summary Studies in animals have determined the importance of specific receptors to the action of many hormones and drugs. In man, a non-invasive external counting technique has been used and absence of receptor function has been demonstrated in a patient with nephrogenic diabetes insipidus using radioactively labelled arginine vasopressin. This is in contrast to the findings in a patient with pituitary diabetes insipidus and a normal control. These results suggest a model for the study of hormone and drug kinetics in man avoiding multiple samplings of biological fluids.

Exposure to ionizing radiation produces several systemic and local reactions which could be mediated by prostaglandins. Prostaglandin levels were therefore studied in blood and tissues of mice which had been exposed to x-rays. Significant increases were found in spleens after 200 to 700 R, and in lungs after 600 to 700 R. These changes were most pronounced 4-7 days after irradiation. Ionizing radiation promptly and potently reduced the activity of prostaglandin dehydrogenase in the spleen, whereas prostaglandin synthesis was less affected. Evidence was obtained for the activation and consumption of haemolytic complement in serum in the course of heart-lung operations involving extracorporeal circulation. Activation involved primarily the classical pathway, and only slightly the alternate pathway.

SUMMARY. Antibodies specific for rubella virion polypeptide, VPI, secreted by clones of hybridoma cells or produced in rabbits in response to specific antigenic stimulation, located determinants for haemagglutination inhibiting (HI) and neutralising (Nt) antibodies on this envelope component.

Modern transfusion practice is associated with an increased risk of transmitting viral agents because of the changing nature of the patients and of the therapeutic blood products. More immunosuppressed patients are receiving blood released faster and with more elaborate blood components. In addition to the classically recognized importance of hepatitis B virus (itself disseminated most efficiently by contamination of products derived from large pools of plasma containing many donations) other agents are assuming increasing importance. They frequently display one or more of the predisposing characteristics of prolonged viraemia, inapparent infections and a carrier or latent state. Some of these infections like cytomegalovirus and the human T-cell leukaemia virus are transmitted only by the cellular component of blood. Others like B and non-A, non-B hepatitis and the putative agent(s) of the newly recognized acquired immune deficiency syndrome can also be transmitted in the plasma or its products. Not all the agents transmitted cause severe illness, however; human parvovirus appears to cause no clinical illness when transmitted by transfusion and infections with non-A, non-B hepatitis are largely detected only by elevations in transaminase levels. Screening tests for the presence of these agents in donor blood or for evidence of infection by them in donors continue to be studied. Other approaches, related in particular to the selection of donors, are becoming increasingly important where serological screening tests are not available.

The aetiology of acute anterior uveitis (AAU) in the great majority of cases is unclear. Various infective agents have been postulated, however, as playing a direct or indirect part in the disease process, possibly via an immune-complex vasculitis. In an earlier study we concluded that, contrary to a previous report, hepatitis B virus (HBV) played only a small role, if any, in the pathogenesis of uveitis in Great Britain. Since we did not look for other markers of HBV infection other than hepatitis B surface antigen (HBsAg), it is possible that some cases of recent HBV infection may have been missed. In the present study 49 patients with AAU were tested for markers of hepatitis B infection. Sera from all patients tested were found to be negative for HBsAg and for antibody to the HBV core (anti-HBc), excluding HBV as a significant cause of AAU in this country. Since the quest for an infective agent in AAU may remain unproductive, future research on the aetiopathogenesis of AAU should include the study of tissue 'vulnerability' and the local and systemic immunoregulatory factors which modulate inflammatory responses.

A renal transplant patient was infected by a transfusion of blood from a chronic carrier of hepatitis B and he also became a symptomless carrier. Stored sera enabled detailed retrospective measurement of the rate of decline of passively transferred HBsAg, anti-HBc, and anti-HBe. Active HBsAg production was detected after two months and anti-HBc after six months; neither HBe nor anti-HBe was actively produced although there were many 42 nm HBV particles in the patient's serum.

The sequences encoding the p24 core protein of human immunodeficiency virus type 1 were identified in a cDNA library made from infected CEM cells. The nucleotide sequence of the DNA coding for p24 was shown to be very similar but not identical to the sequences of lymphadenopathy virus and human T-cell leukaemia virus type IIIb. These sequences were expressed in Escherichia coli at the amino terminus of beta-galactosidase and used to screen a panel of monoclonal antibodies raised against virus-expressed p24. Regions containing the epitopes of five of the monoclonal antibodies were located using a series of amino- and carboxy-terminal deletion mutants of the recombinant p24 protein.

SummaryMice were immunized by three intraperitoneal and one intravenous injection of rubella haemagglutinin. Splenocytes from these mice were fused with the cells of a syngeneic myeloma cell line, and following culture for various periods of time, single-cell clones were derived by the technique of limiting dilution.A total of 139 clones were derived from 13 parent hybrid cultures. To date, four of these cloned cultures have been propagated as ascitic tumours in mice. The preparation of IgG from ascitic fluid and labelling of this antibody with 125I is described. Results indicate that the use of labelled monoclonal antibodies as indicator reagents in solid-phase IgM antibody capture assays for the detection of rubella-specific IgM results in enhanced performance of these tests.

An anti-HIV 1 competitive ELISA was developed using a monoclonal anti-HIV 1 gag to capture viral antigen to the solid phase. This format of assay was compared with a competitive ELISA where a polyclonal human anti-HIV 1 was used, to capture the viral antigen. Several benefits were observed using the monoclonal-antibody form of the assay. Firstly, less viral antigen was needed on solid phase to give an equivalent test in terms of positive and negative optical density 450 nm values. Secondly, a slight increase in sensitivity was also gained without any loss of specificity and finally, as a result of the murine nature of the antibody on the solid phase, no cross-reaction with the labelled human antibody in the test conjugate was observed. Such cross-linking has been observed with the polyclonal form of the assay and can lead to false-negative reactions.

A panel of mouse hybridomas secreting monoclonal antibody to serum hepatitis Be antigen (HBeAg) was produced from mice immunized with denatured hepatitis B core antigen (HBcAg). This panel could be divided broadly into two groups. Within each group, the monoclonal antibodies recognized a single antigenic site, designated either e-alpha or e-beta, and generally exhibited a high degree of cross-inhibition. In contrast, between the two groups of antibodies there was little or no cross-inhibition. The antigens of serum HBeAg and denatured HBcAg appeared to be very similar. Both behaved as molecules carrying only a single e-alpha or e-beta site, in spite of native serum HBeAg having an apparent molecular weight of 300 000. It is inferred that e-alpha and e-beta sites may be involved in the polymerization of HBeAg into HBcAg and that during this process mutual masking of antigenic sites may occur.

SummaryRadial haemolysis (RH) was used to test sera for immunity to rubella from 1317 patients attending a general practice. One hundred and forty-one (10·7%) were treated as susceptible and offered an attenuated virus vaccine (RA 27/3). Pre-immunization sera from 43% of these patients were reactive at low levels in RH (< 15 international units rubella antibody per ml).Pre- (S1) and post- (S2) immunization sera from 66 vaccinees were studied in detail. Antibody was detected by RH, haemagglutination inhibition (HI) and enzyme-linked immunosorbent assay (ELISA), and the specific IgM response was measured by a solid-phase M-antibody capture radioimmunoassay (MACRIA). The vaccine-induced IgM response was only detected if the S1 serum was non-reactive by all tests for rubella antibody. It was weaker than that seen following wild virus infection. It could be detected reliably for six weeks, and in most cases for nine weeks, after immunization. In contrast, patients with S1 specimens reactive by RH, HI or ELISA never showed an IgM response in the S2 specimen despite ‘significant’ antibody rises often being present.It was considered that an IgM response to RA 27/3 was the best indicator of pre-immunization susceptibility to rubella. The failure of many vaccinees to make an IgM response implied that a significant proportion were already immune. It is suggested that the threshold for a report of immunity to rubella could be lowered from 15 i.u. antibody per ml and so fewer women immunized without vaccine being withheld from those who need it.

By November 7, 1983, 24 cases of AIDS in the United Kingdom had been reported to the Communicable Disease Surveillance Centre. At the same time an increasing number of homosexual men with unexplained lymphadenopathy syndrome (LAS) have been seen in our department. Between December 1982 and July 1983, 14 homosexual men with LAS and 11 healthy homosexual men were studied. Patients with LAS had a high number of lifetime episodes of sexually transmitted diseases, a history of recent sexual activity in the United States (9 of 14), sexual contact with British AIDS patients or other persons with LAS (7 of 14), and hypergammaglobulinemia. Low T-helper/T-suppressor ratios (less than 0.8), due mainly to a decrease in T-helper cells, were found in both groups. Lymph node biopsies showed follicular hyperplasia and hypocellular pattern. All 25 patients studied had antibodies to Epstein-Barr virus capsid antigen (anti-VCA) and 11 had antibodies to early antigen (anti-EA); 13 of 17 were excreting the virus; and two showed no Epstein-Barr-virus-specific regression. Peripheral blood immunoglobulin-producing B-cells from six patients with hypergammaglobulinemia were negative for the Epstein-Barr virus nuclear antigen (EBNA). Five lymph node biopsies showed no EBNA-positive cells. Epstein-Barr virus reactivation is common in the patients with LAS and healthy homosexual men in London, but would not seem to be the cause of the polyclonal B-cell activation or lymphadenopathy.

SUMMARYAn M-antibody capture radioimmunoassay (MACRIA) for anti-rubella IgM was developed. Under optimum conditions positive serum specimens bound up to 20 times as much radioactivity as negative specimens. Positive reactions were expressed in arbitrary units/ml by comparison with a calibration curve derived from results obtained with dilutions of a standard serum.The specificity of the assay was confirmed by testing IgM and IgG rich fractions of positive sera. One hundred and forty specimens from blood donors, patients whose sera contained rheumatoid factor and patients with acute, non-rubella, virus infections were tested by MACRIA. No significant non-specific reactions were detected.Paired sera from acute rubella (25 patients) and individual sera from suspected rubella (69 patients) were tested for anti-rubella IgM by MACRIA and by haemagglutination inhibition following sucrose-density-gradient fractionation. There was close agreement between the two methods. The capture assay was more sensitive and could be used to detect the weak IgM response in women given RA 27/3 vaccine. After the natural infection, the MACRIA was strongly positive for two months and remained weakly so for a further two months. Repeat testing of sera demonstrated good reproducibility of the assay.MACRIA proved a simple, sensitive and specific test for anti-rubella IgM and compared favourably with currently used techniques.

Serological markers for hepatitis B virus infection have been examined in 34 patients with acute hepatitis B, 17 of whom developed fulminant hepatic failure. Hepatitis B surface antigen concentrations were significantly lower and hepatitis Be antigen was less frequently detectable in patients with fulminant hepatic failure compared with those with acute hepatitis (median 0.64 micrograms, range 16-0 and median 32 micrograms and range 100-4 micrograms respectively, p less than 0.001; HBeAg detected in 12% and 88% respectively, p less than 0.001). The IgM component of hepatitis B core antibody was significantly higher in the patients with fulminant hepatic failure with median values of 500 IU/ml compared with those with uncomplicated hepatitis (median 202 IU/ml, p less than 0.05 Wilcoxon's rank test). Three patients who developed a fulminant course had detectable levels of either anti-HBs or anti-HBe. These results are consistent with enhanced antibody responses to all three hepatitis B virus antigens and more rapid clearance of the latter during fulminant hepatic failure.